As nutrients become limiting in developing yeast colonies, at least 3 different cell differentiation programs can be induced: 1) pseudohyphal differentiation (a foraging response), 2) sporulation, and 3) programmed cell death. Intriguingly, a key aspect determining an individual cell's choice of fate within a colony is geographical; different regions of the colony adopt different fates. Indeed, we recently discovered that sharp boundaries form between layers of sporulated and unsporulated cells in colonies. Pattern formation within yeast communities likely provides functional advantages over unpatterns communities. The long-range goal of the Honigberg lab is to identify the mechanisms that regulate the self- organization of yeast colonies from homogeneous to highly patterned communities. Given the sharp boundaries between the colonies regions mentioned above, a key part of these mechanisms is likely cell-to- cell signaling. The central hypothesis of the current proposal is that a layer of cells in colonies near the agar surface undergoes apoptotic programmed cell death (PCD), providing nutrients or other signals the stimulate sporulation in an overlying layer of cells. Three complementary approaches are taken to address the above hypothesis. The first approach is to determine the activity of signaling pathways and cellular processes as colonies develop. Specifically, we will suspend colonies and assay the suspended cells using molecular approaches such as protein phosphorylation assays or and cytological approaches such as monitoring apoptotic markers. The second approach is to section colonies to investigate spatial patterns of gene expression, protein localization and cell differentiation. The last approach is to monitor cell fates in communities containing mixtures of strains of different genotypes to test hypotheses regarding the cell autonomy required for PCD, sporulation and patterning in colonies. One of the most ancient and fundamental purposes for communication between organisms may have been to allow these microbes to self-organize into functional communities. Yeast colonies provide an opportunity to investigate this type of communication. In addition to the scientific interest, the health relevance of the proposed research derives from the fact that organized communities of pathogenic yeast termed biofilms can form on implanted medical devices, and that the organization of these biofilms contributes significantly to the lethality of hospital-acquired fungal infections.